Cell slides are ordinarily prepared during a cytopathology procedure, i.e. studying and diagnosing diseases on the cellular level. One of the most prevalent methods of slide preparation is smearing. The samples are smeared across a glass microscope slide for subsequent staining and microscopic examination. The smearing method is required in order to acquire a thin layer of cells on the slide, thus enabling focusing on and imaging the cells. However, smearing might cause a change in cell morphology. In addition, with smearing it is difficult to accurately and stably image living cells, for at least the reason that cells dry out quickly and additional staining without fixation is almost impossible.
Anthony Moody describes [Various rapid diagnostic tests for Malaria parasite in Clinical Microbiology Reviews January 2002 p. 66-78] inter alia, preparation of thin blood films containing a monolayer of red blood cells and multilayered thick blood films.
Reference to Moody is later made by Vink J P. et al. [An automatic vision-based malaria diagnosis system Journal of Microscopy, 2013, p. 1-13] which describes a quantitative cartridge-scanner system for vision-based malaria diagnosis, focusing on low malaria parasite densities. The proposed cartridge allows the formation of a thin blood film and detection of Plasmodium falciparum. To be able to determine the parasite density, Vink et al. aimed at forming a thin blood film containing a monolayer of red blood cell and based their design on the cartridge described in International patent application publication No. WO 2010/116341 (US patent application publication No. 20120225446).
Specifically, WO 2010/116341 describes an apparatus for producing thin layers of a fluid sample for analysis, that has a two dimensional array of analysis chambers, and a branching pattern of entry channels coupled to the array to enable the analysis chambers to be filled in parallel. The analysis chambers are planar with a height less than that of the entry channels so as to produce the thin layers when filled with the fluid sample. The analysis chambers can be suitable for capillary filling by a specified fluid sample such as blood. The analysis chambers should not be more than 15 μm high in order for the cells to form a monolayer. Manufacturing of chambers having height of this order is not always possible and is relatively expensive. U.S. Pat. No. 4,209,548 describes a method wherein a blood sample on a slide is spun to create a monolayer of randomly distributed red blood cells. To inhibit cell morphology distortion from occurring during drying, the morphologies of the cells contained in the monolayer are preserved by a fixing agent after monolayer preparation but prior to drying. U.S. Pat. No. 4,494,479 describes a device for preparing a monolayer film of a biological fluid sample on a slide device that includes a base for retaining a slide thereon and a spreader manually movable linearly relative to the base and slide in a pass which spreads a sample of the fluid on the slide into such a monolayer.
The publication “Counting blood cells with countess Automated Cell Counter” describes preparation of blood samples for counting white/red blood cells that involves dilution of the blood cells.
U.S. Pat. No. 6,819,408 describes a method and apparatus for analyzing a blood or another biological fluid sample in a quiescent state without the need for additional diluting reagents or fluid streams passing through the apparatus during the analytic process. The method and apparatus allow enumeration of particulate constituents of biological samples and inspection thereof using an optical scanning instrument.
Leif R C et al (Methods for Preparing Sorted Cells as Monolayer Specimens) Springer Lab Manuals 2000 describes the application of a method of centrifugal cytology for creating a monolayer from cells that were previously sorted using a cell sorter (FACS). According to Lief, Centrifugal Cytology is the process where cells in suspension are centrifuged onto a substrate and then fixed concurrently with the application of centrifugal force.
Knessel E A et al (Roche Image Analysis Systems) Acta Cytologica 1996 describes the application of ma a batch centrifugation process together with a computer controlled robotic pipetting station to prepared a monolayer from a suspension of cervical sample.
Zahniser D J et al. (Automated Slide Preparation System for Clinical Laboratory) Cytometry 1996, describes an automated device that collects cells from suspension and disperses them as a monolayer on a glass slide using filter-transfer technology.
General Description
The present disclosure provides a method for imaging a blood sample, the method comprising:                introducing a cell suspension comprising red blood cells, onto a base surface of a carrier having a vertical height (H) being greater than or equal to a vertical depth (h) of said cell suspension when on said base carrier, the cell suspension comprising a cell concentration (C) being determined by the function:C=F/(h*pi/4*d2)        (F) being a desired base surface coverage; and (d) being an average cell dimension of the cells in the cell suspension;        allowing the cells in the cell suspension to settle on said base surface of the carrier to form on the base surface of the carrier a monolayer of cells;        acquiring at least one microscope image of at least a portion of the monolayer of cells,        
wherein said at least one microscope image is obtained by a microscope set to Depth Of Field (DOF) that is not more than 20% of the vertical height of the cell suspension when settled on said base surface.
Also provided by the present disclosure, a kit for imaging a blood sample, the kit comprising:                a carrier comprising a base surface and a vertical height (H); and        instructions for performing the steps of:                    providing a cell suspension from a blood sample comprising red blood cells, the cell suspension being of a cell concentration (C) determined by the function:C=F/(h*pi/4*d2)                        (F) being a desired base surface coverage; and (d) being an average cell dimension of the cells in the cell suspension;        introducing the cell suspension of the desired concentration C onto the base surface of the carrier, the cell suspension having said vertical depth (h) when in said carrier, said vertical depth (h) being smaller or equal to the vertical height (H);        allowing the cells in the cell suspension to settle on said base surface of the carrier to form onto the base surface a monolayer of cells;        acquiring at least one microscope image of at least a portion of the monolayer of cells,        wherein said at least one microscope image is obtained by setting the microscope to a Depth Of Field (DOF) that is not more than 20% of the vertical height of the cell suspension when settled on said base surface.        
Yet further, there is provided by the present disclosure a system for imaging a blood sample comprising:                one or more reservoir units for holding, respectively, one or more sample treatment agents comprising at least one blood cells diluting agent;        a blood sample preparing unit being in fluid communication with said one or more reservoir units and configured to receive a blood sample comprising red blood cells and amount of at least one blood cell diluting agent and to form therefrom a blood cells suspension, the amount of said at least one cell diluting agent being determined so as to dilute said sample of cells by a dilution factor (D) so as to provide a cell concentration (C);        a microscope image acquisition unit for acquiring at least one image of the blood cells suspension when on a base surface of a carrier, the carrier having a vertical height (H) being greater or equal to a vertical depth (h) of said cell suspension when on said base surface;        a control unit being configured to:                    provide dilution factor D of diluting said sample, factor D being a function of the desired base surface coverage (F), the average cell dimension d of cell blood cells, and the vertical depth h of said suspension of cells that provides a monolayer of the cells when settled on said base surface of the carrier; and            acquire at least one microscope image of the cell suspension by a microscope set to a Depth Of Field (DOF) that is not more than 20% of the vertical height of the cells suspension when settled on said base surface.                        